C-peptide is the linking peptide between the A- and B-chains in the proinsulin molecule. After cleavage in the endoplasmic reticulum of pancreatic islet β-cells, insulin and a 35 amino acid peptide are generated. The latter is processed to the 31 amino acid peptide, C-peptide, by enzymatic removal of two basic residues on either side of the molecule. C-peptide is co-secreted with insulin in equimolar amounts from the pancreatic islet β-cells into the portal circulation. Besides its contribution to the folding of the two-chain insulin structure, further biologic activity of C-peptide was questioned for many years after its discovery.
Type 1 diabetes, or insulin-dependent diabetes mellitus, is generally characterized by insulin and C-peptide deficiency, due to an autoimmune destruction of the pancreatic islet 3-cells. The patients are therefore dependent on exogenous insulin to sustain life. Several factors may be of importance for the pathogenesis of the disease, e.g., genetic background, environmental factors, and an aggressive autoimmune reaction following a temporary infection (Akerblom H K et al.: Annual Medicine 29(5): 383-385, (1997)). Currently insulin-dependent diabetics are provided with exogenous insulin which has been separated from the C-peptide, and thus do not receive exogenous C-peptide therapy. By contrast most type 2 diabetics initially still produce both insulin and C-peptide endogenously, but are generally characterized by insulin resistance in skeletal muscle and adipose tissue.
In addition to type 1 and type 2 diabetics, there is increasing recognition of a subclass of diabetes referred to as Latent Autoimmune Diabetes in the Adult (LADA) or Late-onset Autoimmune Diabetes of Adulthood, or “Slow Onset Type 1” diabetes, and sometimes also “Type 1.5” or “Type one-and-a-half” diabetes. In this disorder, diabetes onset generally occurs in ages 35 and older, and antibodies against components of the insulin-producing cells are always present, demonstrating that autoimmune activity is an important feature of LADA. It is primarily antibodies against glutamic acid decarboxylase (GAD) that are found. Some LADA patients show a phenotype similar to that of type 2 patients with increased body mass index (BMI) or obesity, insulin resistance, and abnormal blood lipids. Genetic features of LADA are similar to those for both type 1 and type 2 diabetes. During the first 6-12 months after debut the patients may not require insulin administration and they are able to maintain relative normoglycemia via dietary modification and/or oral anti-diabetic medication. However, eventually all patients become insulin dependent, probably as a consequence of progressive autoimmune activity leading to gradual destruction of the pancreatic islet 3-cells. At this stage the LADA patients show low or absent levels of endogenous insulin and C-peptide, and they are prone to develop long-term complications of diabetes involving the peripheral nerves, the kidneys, or the eyes similar to type 1 diabetes patients and thus become candidates for C-peptide therapy (Palmer et al.: Diabetes 54(suppl 2): S62-67, (2005); Desai et al.: Diabetic Medicine 25(suppl 2): 30-34, (2008); Fourlanos et al.: Diabetologia 48: 2206-2212, (2005)).
Type 1 diabetics suffer from a constellation of long-term complications of diabetes that are in many cases more severe and widespread than in type 2 diabetes. Specifically, e.g., microvascular complications involving retina, kidneys, and nerves are a major cause of morbidity and mortality in patients with type 1 diabetes.
Roughly 30% of all patients with type 1 diabetes develop diabetic nephropathy characterized by gradually increasing albuminuria, decline in glomerular filtration rate (GFR), and elevated systemic blood pressure 10 to 20 years after the onset of the disease. Five years later many of these patients may suffer from end-stage renal disease (Bretzel R G: J. Diabetes Complications 11(2): 112-122, (1997)); Chiarelli F et al.: Annual Medicine 29(5): 439-445, (1997)), a condition requiring hemodialysis or transplantation. Microalbuminuria, defined as a urinary albumin excretion rate between 30 and 300 mg/day, strongly predicts the development of nephropathy in diabetes. The diabetes patients with nephropathy suffer a high mortality rate compared with diabetes patients without nephropathy.
A multiplicity of peripheral nerve dysfunctions may develop in approximately 50% of the patients with type 1 diabetes (Dyck P J et al.: Neurology 43(4): 817-824, (1993)). The distal symmetric polyneuropathy that predominantly affects sensory and autonomic function is the most common manifestation (Fedele D et al.: Drugs 54(3): 414-421, (1997)) with symptoms such as paresthesias, numbness, and hyperalgesia and in some cases severe pain. Typically, the symptoms initially occur in the lower limbs. Diabetic neuropathy is the leading reason for limb amputation.
Diabetic eye disease and its complications, especially diabetic retinopathy, are leading causes of blindness and visual dysfunction in developed countries. Approximately 25% of the patients with type 1 diabetes have retinopathy after five years of the disease, increasing to 60% and 80% after 10 and 15 years, respectively (Aiello L P et al.: Diabetics Care 1(1): 143-56, (1998)).
There is currently no causal treatment for prevention of long-term complications in patients with diabetes. However, maintenance of glucose concentrations at the near-normal level is of utmost importance, as it may to some degree delay the onset and retard the progression of diabetic nephropathy, neuropathy, and retinopathy (DCCT Research Group: New England Journal of Medicine 329: 977-86, (1993)).
There is increasing support for the concept that C-peptide deficiency may play a role in the development of the long-term complications of insulin-dependent diabetics. Additionally, in vivo as well as in vitro studies, in diabetic animal models and in patients with type 1 diabetes, demonstrate that C-peptide possesses hormonal activity (Wahren J et al.: American Journal of Physiology 278: E759-E768, (2000); Wahren J et al.: In International textbook of diabetes mellitus Ferranninni E, Zimmet P, De Fronzo R A, Keen H, Eds. Chichester, John Wiley & Sons, (2004), p. 165-182). Thus, C-peptide used as a complement to regular insulin therapy may provide an effective approach to the management of type 1 diabetes long-term complications.
Studies to date suggest that C-peptide's therapeutic activity involves the binding of C-peptide to a G-protein-coupled membrane receptor, activation of Ca2+-dependent intracellular signalling pathways, and phosphorylation of the MAP-kinase system, eliciting increased activities of both Na+, K+-ATPase and eNOS. Additionally, some studies indicate that C-peptide may interact with insulin, an effect that may be mediated, at least in part, by the dispersal of zinc insulin hexamers via C-peptide. Furthermore, the simultaneous subcutaneous (S.C.) injection of hexameric insulin and C-peptide at the same injection site, but not different injection sites, results in increased glucose utilization, a more pronounced antilipolytic effect, and a more marked depression of plasma glucagon levels in type 1 diabetic patients compared to administration of insulin alone (WO 2007/015069, entitled “Compositions and methods of treating diabetes”; Shafqat et al.: Cell. Mol. Life. Sci. 63: 1805-1811, (2006)). It is thus suggested that C-peptide plays a role in promoting the disaggregation of insulin hexamers both in vivo, and when co-administered with insulin at the same site.
The estimated association constant for C-peptide binding to its cellular receptor is 3×109 M−1. Half-saturation of C-peptide binding to renal tubular cells occurs at approximately 0.3 nM (Rigler R et al.: PNAS USA 96: 13318-13323, (1999)). Consequently, it is likely that in healthy humans near-saturation of the receptor is reached at normal circulating levels of C-peptide which vary from about 0.47±0.15 nM; (range 0.19 to 0.99 nM) when fasting, to about 2.51±0.75 nM; (range 0.72 to 6.5 nM) postprandially (Dalla-Man et al., Diabetes 54: 3265-3273, (2005)). This finding helps explain the consistent observation that effects of C-peptide cannot be demonstrated in non-diabetic animals or healthy subjects (Hoogwerf B et al.: Metabolism 35: 122-125, (1986); Johansson B L et al.: Diabetologia 35: 1151-1158, (1992); Wojcikowski C et al.: Diabetologia 25: 288-290, (1983)); it is only in diabetic animals or in patients with C-peptide deficiency that specific effects of C-peptide have been observed.
Studies of type 1 diabetes in animal models demonstrate that C-peptide in replacement doses has the ability to improve peripheral nerve function and prevent or reverse the development of nerve structural changes. Thus, C-peptide administration for 2 months (75 nmol/kg/24 h S.C. using osmotic pumps) in diabetic BB/Wor rats, starting one week after onset of diabetes, reduces the development of the acute nerve conduction velocity (NCV, sciatic-tibial nerve) defect by 60% compared to non-replaced diabetic control animals (Sima A et al.: Diabetologia 44: 889-897, (2001)).
In a short-term study, involving human C-peptide infusion (0.5 nmol/kg/min intravenous [I.V.]) for 60 min in STZ-D rats, it could be demonstrated that compared to control animals, glomerular hyperfiltration decreased, albumin excretion fell and renal functional reserve rose (Sjöquist M et al.: Kidney International 54: 758-764, (1998)).
Over the last 15 years at least 19 clinical trials have been performed on C-peptide in humans, including approximately 340 type 1 diabetes patients receiving recombinantly produced C-peptide. C-peptide has been administered I.V. in doses of 5-30 pmol/kg/min for one to three hours and S.C. in single doses of 60-1800 nmol (approximately 0.18 to 5.4 mg) or in repeated injection in a total dose of 600-3,000 nmol/day (approximately 1.8 to 9 mg/day) S.C. given 3-4 times daily for three months. In one study C-peptide was administered together with insulin via a pump for one month (Johansson B L et al.: J. Clin. Endocrin. Metab. 77(4): 976-981, (1993)). Most of the studies were randomized, double blind, and placebo controlled. The studies focused on vascular dysfunction, metabolic effects and kinetics, and early-stage nephropathy, neuropathy, and retinopathy. Several physiological effects of C-peptide were observed in diabetes patients lacking endogenous C-peptide production. (Ekberg K et al.: Diabetes 55: 536-541, (2003); Hansen A et al.: Diabetes 51: 3077-3082, (2002); Johansson B L et al.: Am. J. Phys. Endocrin. Metab. 285: E864-E870, (2004); Johansson B L et al.: Diabetic Med. 17(3): 181-189, (2000); Fernqist-Forbes E et al.: Acta Physiol. Scand. 172: 159-165, (2001); Johansson B L et al.: Acta Physiol. Scand. 165: 39-44, (1999); Johansson B L et al.: Diabetologia 39(6): 687-695, (1996); Oskarsson P et al.: Diabetic Med. 14: 655-659, (1997); Johansson B L et al.: Diabetologia 35: 1151-1158, (1992); Sjoberg S et al.: Diabetologia 34: 423-428, (1991); Johansson B L et al.: Diabetologia 35: 121-128, (1992)).
Despite these intensive efforts, and long-felt need for an effective therapy for the treatment of the long-term complications of insulin-dependent diabetes, C-peptide has yet to be approved for therapeutic use. A significant barrier to the development of a commercially viable C-peptide therapy lies in the need to establish an effective dosing regimen for C-peptide and insulin administration that is both therapeutically effective in preventing or reversing the effects of the long-term complications of insulin-dependent diabetes, and safe and well tolerated by the patient.
The present invention is focused on the development of more effective C-peptide therapies for the treatment of the long-term complications of diabetes. In one aspect, such improved therapies are based on improved dosing regimens for C-peptide and insulin that reduce the risk of the patient developing hypoglycemia, while maintaining good glycemic control, and/or avoiding excessive weight gain. These improved methods and kits are based on clinical trial results that surprisingly demonstrate that subcutaneous C-peptide administration in a subset of patients with neuropathy results in a sustained reduction in insulin requirements, and therefore confers an increased risk of hypoglycemia in such patients. The present invention is further based in part on the discovery that the risk of hypoglycemia in such patients can be mitigated while maintaining good glycemic control by reducing the patient's insulin dose during on-going C-peptide therapy. In one aspect, such improved dosing regimens are based on an assessment of the response of the patient to C-peptide administration.